Process for obtaining a substance lowering the blood pressure



Patented Feb. 13, ,1 940 PROCESS FOR OBTAINING LOWERING THE BLOOD Ernst Wollheim, Berlin-Oharlottenburg,

A SUBSTANCE raessuas Germany No Drawing. Application November 18, 1936,

Serial No. 1935 111,557. In Germany November 21,

10 Claims. (Cl. 167-74) My invention relates to-a process for obtaining a substance lowering the blood pressure from human or animal urine.. The obtained substance has the special property of being resistant to boiling. Hence, by boiling, it is possible to readily separate it from other active substances or hormones contained in the urine but not resistant to boiling. ecuted 1n thepreparation of the substance lowering the blood pressure for destroying the accompanying substances not resistant ,to boiling may be efiected as well in an acid as in a neutral medium, but also alkaline reaction.

I have found that blood pressure may be obtained fromthe urine most readily and quantitatively, if the urine is treated with water soluble sulphates, preferably .with sulphate of ammonia, before'or after boiling and neutralizing; but conveniently after the execution of these operations. Other water-soluble sulphates, such as sodium sulphate, however,

may also be employed for this not fish quite the same high success.

It been proven by my prior researches that all normalurine of men or animals, particularly of mammalia, contains considerable quantities of the sub-stance active in lowering the blood pressure and which substances are resistant to boiling, whereas this substance is missing in the urine .of individuals sick with high blood pressure. I have also found this active substance present in the urine of pregnant women and animals. When working up urine of the latter kind there is only the difiiculty to'be overcome, that the active agent lowering the blood pressure must also be separated from the'ovarial hormone contained therein and which also is resistive against boiling. This separation may be executed by applying such methods as for instance extracting with ether, precipitating with alcohol and the like, by which only one of said components will be dissolved or precipitated, respectively. Whereas the ovarial hormone, as is well known, is

' readily soluble in ether, the active agent lowering the-blood pressure, the preparation of which is here in question, essentially is insoluble in ether. Therefore both substances may be separated froma raw material containing both, for instance by first extracting the boiled urine in neutral or acid form with ether, and then treating the to separate therefrom the ering the blood pressure. For destroying the hormones not resistant to active substance lowhe boiling consequently to be exthe substance lowering the purpose, thoug part not taken'up by the ether in order 2.

ally it is sufiicientto boil boiling and accompanying the. active substance lowering the blood pressure, in the urine, generonce for a relatively short time. But, to make sure, one, may boil for some longer time, for instance for five minutes in neutral or acid solution. E'referably said boiling'is effected before any other treatment of the urine, in order thus to destroy from the first the undesired substances, thereby facilltating and accelerating the concentration or enrichment of the substance .lowering the blood pressure. But, under certain circumstances, one may proceed reversely in first concentrating said substance and then destroying the undesired accompanying substances not resistive against boiling by boiling the resultant concentrated solution. The remaining liquid jected to a furtherrefinement or purification, if desired. In all cases the urine maybe thickened by heating, if desired in a vacuum.

Examples (1) it liters of urine are boiled, neutralized and filtered. In this urine 7 kilos of ammonium sulphate are dissolved. (This gives a solution containing about 29% ammonium sulphate) A fine, rather colourless sediment then separates slowly. The mass is left standing at or below ordinary room temperature during several hours, for instance over night, until the separation is complete, after which it isfiltered 01f The residue is taken up with 700 cubic centimeters of water, and the formed'solution is separated from any,remaining residue. From the aqueous solution obtained the active substance is precipitatedanew by saturating with sulphate of ammonia. Again a white sediment is formed which, when complete, is filtered off as described above and then is dissolved in water. From the again formed solution the sulphate of ammonia is eliminated by any convenient method,.for instance by dialysis or electrodialysis, or the like. This is possiblebecause the active substance lowering the blood pressure is not dialysable. v

If 0.5 to 1 cubic centimeter of the obtained (2) The solution obtained according to Example 1 by'dissolving'in 700 cubicrce'ntimeters of water -the first precipitate obtained bymeans of sulphateof ammonia may be purified further-by treating said solution with five times its quantity of methanol or four hydrous acetone.

may then be subsolution are injected to a rabbit, the blood pres.-

sure of this animal will be lowered very-considerably for about thirty minutes to one hour.

times its quantity of an-, I They active substance is precipitated thereby and is freed from the remaining solution by filtration or by centrifuging. The

obtained solid product is repeatedly washed with ether and with concentrated or absolute alcohol o and is then dissolved again by means of 200 cubic centimeters of water. This solution is. freed from impurities, especiallyfrom salts yet contained therein, by dialysis or electrodialysis during 24 hours, The remaining solution containing the be further concentrated in vacuo, can be kept for a considerable period without objectionable fermentation; and'without loss of activity. I

Instead of -.first t1 7eating the urine with sul- ."phate of ammonia. and thereafter treating the solution methanol or the like, the reverseLordei' by urine with methanol or acetone to precipitate a' material containingtheactive substance, dissolving the. thus obtained precip'tate in water one. may

and then 'precipitating the active substancej solution bymeans of sulphate afresh from this of ammonia.

(3) viii liters of urineare boiled, neutralized and filtered. Then 8. kilos of: sodium sulphate are dissolved in this filtrate.- A considerable quantityo'f. sediment slowly separates. The mass :is left, standingforsome hours, preferably over night, at fordin'ary or reduced temperatureuntil the separation will be completed. The sediment thenis'filtered off and ,washed three times with concentrated. alcohol. and'fethe'r. .Thereafter it I is taken uplwith 600 cubic centimeters of water and shakentherewith during 4 hours, whereupon the formed solution is, separated from any residue remaining; To the.solution thus obtained five times its quantity of methanol orv four .quantity of anhydrous acetone is added,and the further" treatment is performed in the manner indicatedirgExample 2.'

Having pow-particularly described and ascertained .the nature of.;my said invention, I declare 5-.that WhatI claim is:

stance capable of lowering the blood pressure, which comprisesadding a water soluble sulphate tc urineginamountsufi'icient to produce a precipitate containing the desired substance, allowing the mixture to stand'for at least several hours, separating said=;precipitate from-the liquid,

theraf ter dissolving lowering the blood pressure, in water, and at somestage of the process, heating containingthe active-substance toabout the boilingppint. I

2-. A ro stance capable of lowering the;bloodflpressure, which' comprises boiling separating said dissolving the precipitate. g

3. -A process of obtaining-from. urine,-a substance; capable the samezwith a water soluble sulphatein amount sufiicient'to cause preci'itation. of.'such desired substance,-"'and boiling-for a short time, thereby obtained from the :first precipitate with.

also proceedin first treating the boiledtimes its 1. A process for obtaining fromurine, a subthe liquid of obtaining from urine, 5' Sllbthe urine; and-adding thereto ammonium sulphate, thereby forming a precipitate containing. the desired substanc,e',--and' of lowering the blood pressure, whicl1 comprises concentrating urine; treating active substance may be used'as such 'or may if desired. It

' stance capable of, lowering stance in a purer condition and boiling the'Solupregnant female mammal, which comprises boilganic liquid saidsubstance capable of stance capable of lowering the blood 4. A process of obtaining from urine, a sub stance capable of lowering the blood pressure, which comprises boiling the urine, and adding a water soluble sulphate in the solid state thereto, thereby forming a precipitate containing the 5 desired substance, and separating such precipiw tate from the liquid, redissolving such precipitate in water and reprecipitating. whereby the product is purified.

5. A,process for obtaining from the urine of, a

. pregnant mammal, a substance capable of lowering the blood pressure, which comprises boiling the said urine for a short time, then extracting the same with ether, and thereafter treating the same with a water soluble sulphate, thereby forming a precipitate containing the desired substance, and-separating such precipitate from the liquid and dissolving the precipitate in water.

6; A process for obtaining from urine, a subpressure, which comprises boiling the urine, thereafter adding a water. soluble sulphate, thereby forming a precipitate containing the desired substance, dissolving such precipitate in water, adding methanol to .the so obtained aqueous solution,

a precipitate containing. the desired-substance,

then dissolving such' precipitate in water, adding a water soluble sulphate to said aqueous solution,

and thereby again precipitating the desired subtion containing the dissolved active substance at any stage of process.

9. A process for obtaining a substance capable of. lowering the blood pressure, from"' rir e, of a 60 ing the urine, extracting the.urine with an orwhich is immiscible with water and which is a solvent for ovarial. hormone, and thereafter treating the urine with ammonium.

sulphate, .whereby a precipitate is' formed in 55 the aqueous liquid and thereafter separating said of :lowering the. precipitate-'-.from the liquid-and -forming-a precipitate containing .thedesired 1 substance, and separating such precipitate from the-liquid; dissolving the precipitate-and .rem. pre ipitating. the blood-pressure-lowering sub 76 stance from such solution.

which contains'theidesired substance and redissolying such precipithe blood precipitate w from the liquid, tated substance capable of lowering pressure, in water. a 60 10. A process of producing a suhs ce capable 1 blood pressure, which comprises precipitating said substance from urine by adding a sufficient amount of ammonium sulphate to said urine allowing the same to stand for at 6:

least several hours, separating the precipitated material."redissolving the precipitate in'water,

. and reprecipitating said substance, and at some stage of' the treatment, subjecting .a material containing said substanceto a temperature equal 7 tothe boiling point of urine, to decompose material present therewith which is unstable at such temperatures.

, ERNST wopumm. 

